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Polymerase Chain Reaction and Agarose Gel Electrophoresis

This document describes procedures for polymerase chain reaction (PCR) and agarose gel electrophoresis (AGE). PCR is used to amplify DNA templates using primers, DNA polymerase, and thermal cycling. AGE separates and visualizes the amplified DNA fragments by size as they migrate through an agarose gel matrix under an electric field. The DNA bands are then imaged to analyze the results of PCR amplification.

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Edward Hu
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17 views4 pages

Polymerase Chain Reaction and Agarose Gel Electrophoresis

This document describes procedures for polymerase chain reaction (PCR) and agarose gel electrophoresis (AGE). PCR is used to amplify DNA templates using primers, DNA polymerase, and thermal cycling. AGE separates and visualizes the amplified DNA fragments by size as they migrate through an agarose gel matrix under an electric field. The DNA bands are then imaged to analyze the results of PCR amplification.

Uploaded by

Edward Hu
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Laboratory 7: Polymerase Chain Reaction and Agarose Gel Electrophoresis

Objectives
• To amplify DNA using polymerase chain reaction (PCR)
• To visualize the length of the DNA product by agarose gel electrophoresis (AGE)

7.1 PCR

Materials
- DNA template
- Taq DNA polymerase
- 10X reaction buffer
- dNTP mix (dATP, dCTP, dGTP, dTTP)
- 10 μM universal forward primer
- 10 μM reverse primer (100, 200, 500, 1000 bp)
- PCR grade water
- Ice
- Pipettors (P2, P20, P200)
- Pipette tips (10 μL, 200 μL)
- Microcentrifuge rack
- 0.2 mL PCR tubes
- PCR tube rack
- PCR machine
- Centrifuge
- Vortexer

Procedure
1. Thaw out tubes of Taq buffer, dNTP mix, forward primer, reverse primer on a microcentrifuge
rack at room temperature for 5-10 minutes. Once the solutions are thawed out, put the tubes on
ice until needed.
2. Obtain 0.2 mL PCR tubes and set them on a PCR rack.
3. Write on the lids of the tube group number, and on the side of the tubes with your initial and date.
4. Mix all of reagents as follows:

Reaction mixture
(for 1 reaction)
PCR grade water 39 μL
10X reaction buffer 5 μL
dNTP mix 1 μL
10 μM Forward primer 1.5 μL
10 μM Reverse primer 1.5 μL
Taq DNA polymerase 1 μL
DNA template 1 µL
Total volume 50.0 μL

5. The reagents should be added with order from top down. Pipette up and down to mix the mixture
after adding all reagents.
6. Mix the contents by vortexing on the vortex mixer at setting of 5 seconds. Spin the tube in a
microcentrifuge at FULL speed (13,200 rpm) for 10 seconds. Put the tube back on ice.

7. Keep your samples in ice. Your instructor will run the PCR machine for you with the following
cycle information.
a. 95 °C, 5 min
b. 25 cycles of 95 °C for 1 min, 57 °C for 1 min, and 72 °C for 1 min;
c. 72 °C, 5 min;
d. 4 °C, infinity.

CLEAN UP
7.2 AGE

DNA is negative in charge due to its sugar-phosphate backbone. Therefore, DNA will migrate towards
the positive anode in the presence of an electric field. Agarose is a chain of sugar molecules extracted
from seaweed. If we dissolve agarose in boiling water and let it cool down, the sugar chains will cross-
link with each other (a process called polymerization), creating ‘pores’ in between, and finally forms a
semi-solid gel-like matrix. Those ‘pores’ present in agarose gel are very tiny and comparable in size to
DNA molecules. Agarose gel can confer a sieving effect when DNA is passing through. Agarose gel
electrophoresis is a process that separates DNA fragments by applying electricity to cause DNA passing
through an agarose gel. DNA fragments shorter in size will move faster and longer DNA fragments will
lag behind. The distances moved by linear DNA molecules are inversely proportional to the log10 of their
molecular size.

DNA molecule is colourless. A staining step is needed so that it can be visualized on an agarose gel. We
will use gel red, a safer staining dye, in this experiment.

In this experiment, PCR products from last lab session will be used to perform the agarose gel
electrophoresis.

Materials
- PCR product from last lab session
- DNA ladder (containing DNA fragments of known size)
- 10X GelRed
- 6X Gel Loading Buffer
- Agarose powder
- 1X TAE buffer
- 250 ml conical flask
- Electrophoresis unit
- Power pack
- Microwave oven
- Gel imager
- Pipettor (P2 &P20)
- Pipette tips

Procedure
Preparation of agarose gel for electrophoresis
1. Dissolve 0.75 g agarose powder in 50 mL 1X TAE buffer (resulting a 1.5% agarose gel) in a 250 mL
conical flask with a loose-fitting cap. The buffer should not occupy more than 50% of the volume of
the flask.
2. Heat the slurry in a microwave oven (or a hot plate) for 2 minutes until the agarose dissolves.
(Caution: the agarose solution can become superheated and may boil violently under prolonged
heating.)
3. Cool the solution for 1 minute. Pour the gel immediately into the plastic tray placed horizontally on
the bench. Insert the comb and check for the presence of air bubbles under or between teeth of the
comb.
4. Allow the gel to set at room temperature for at least 20 minutes.

Gel Electrophoresis of DNA


1. After the gel is completely set, remove the comb and sealing tape carefully, and mount the gel in the
electrophoresis tank.
2. Add an appropriate amount of 1X TAE buffer to cover the gel to a depth of about 1 mm. (Note: It is
important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the
gel.)
3. Pipette 20 μL of the PCR product into 0.2 mL PCR tube
4. Pipette 2 μL of 10X GelRed to the PCR tube from Step 7 and fully mix them with vortex.
(Remarks: 2 μL / (2+20) μL, 10X GelRed is diluted to 1X)
5. Pipette 4.4 μL of 6X Gel Loading Buffer into the PCR tube from Step 8. (Remarks: 4.4 μL /
(22+4.4) μL, 6X Gel Loading Buffer is diluted to 1X)
6. Pipette 2 μL of 10X GelRed into 18 μL DNA ladder and pipette up and down to mix it or using
vortex. (Remarks: this process is also diluting 10X GelRed to 1X. i.e. 2uL / (2uL+18uL))
7. Slowly load 5 μL of stained DNA ladder (from Step 11) at the first well of the gel.
8. Slowly load 5 μL stained DNA samples (from Step 10) into wells of the gel.
9. Close the lid of the gel tank and attach the electrical leads so that the DNA (DNA is negatively
charged) will migrate toward the anode (red lead, positive terminal).
10. Apply a voltage of about 120 V. If leads have been attached correctly, bubbles should be generated
at the electrodes.
11. Run the gel for 20 min.
12. Turn off the electric current and remove the gel from the gel tank.
13. Take a picture of the gel in gel imager to visualize the DNA bands.

CLEAN UP

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